Monocyte chemoattractant protein-1 expression by osteoblasts following infection with Staphylococcus aureus or Salmonella.

Two common pathogens of bone, Staphylococcus aureus and Salmonella, were investigated for their ability to induce chemokine expression in bone-forming osteoblasts. Cultured mouse or human osteoblasts could rapidly respond to bacterial infection by upregulating the mRNA encoding the chemokine, monocyte chemoattractant protein-1 (MCP-1). This rapid induction occurred on infection with either the gram-positive pathogen, S. aureus, or the gram-negative pathogen, Salmonella. Increased mRNA expression translated into MCP-1 secretion by cultured mouse or human osteoblasts in response to viable bacteria, whereas UV-killed bacteria were less effective in stimulating chemokine secretion. There was a dose-response relationship observed between the amount of input bacteria and increases in MCP-1 secretion. Immunohistochemical staining of infected osteoblasts indicated that the majority of cells could express MCP-1, with some osteoblasts having a higher intensity of staining than others. Organ cultures of mouse calvaria (skullcap) bone showed increases in MCP-1 immunostaining following bacterial infection. The immunoreactive MCP-1 in infected calvaria localized to areas containing active osteoblasts. Taken together, these studies demonstrate a conserved osteoblast-derived MCP-1 response to two very different pathogens of bone.     

Staphylococcus aureus and Salmonella enterica serovar Dublin induce tumor necrosis factor-related apoptosis-inducing ligand expression by normal mouse and human osteoblasts

Staphylococcus aureus and Salmonella enterica serovar Dublin invade osteoblasts and are causative agents of human bone disease. In the present study, we examined the ability of S. aureus and Salmonella serovar Dublin to induce the production of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by normal osteoblasts. Normal mouse and human osteoblasts were cocultured with S. aureus or Salmonella serovar Dublin at different multiplicities of infection. Following initial incubation and examination of TRAIL expression, extracellular bacteria were killed by the addition of media containing the antibiotic gentamicin. Lysates and conditioned media from osteoblast cultures were then collected at various times following invasion and analyzed. The results demonstrated that S. aureus and Salmonella serovar Dublin are potent inducers of TRAIL expression by osteoblasts. Mouse and human TRAIL mRNA expression was induced by bacterial infection and demonstrated a dose-dependent response. Analysis of kinetics suggested that TRAIL mRNA was induced within 30 min after exposure to bacteria and that its level of expression remained relatively constant over the time period examined. mRNA molecules encoding TRAIL receptors were constitutively expressed by osteoblasts. Furthermore, TRAIL protein was detected as early as 45 min and up to 24 h following infection. The quantity of TRAIL protein produced also increased in a dose-dependent manner. Collectively, these findings suggest a mechanism whereby bacterial pathogens mediate bone destruction via osteoblast apoptosis.     

Functional CD40 expression induced following bacterial infection of mouse and human osteoblasts

Bacterially induced bone infections often result in significant local inflammatory responses which are coupled with loss of bone. However, the mechanisms necessary for the protective host response, or those responsible for pathogen-induced bone loss, are not clear. Recent evidence demonstrates that bacterially infected osteoblasts secrete chemokines and cytokines, suggesting that these cells may have an unappreciated role in supporting localized inflammation. In this study, mouse and human osteoblasts were investigated for their ability to express functional CD40 upon exposure to two important pathogens of bone, Staphylococcus aureus and Salmonella enterica serovar Dublin. Bacterial infection of cultured mouse or human osteoblasts resulted in increased CD40 mRNA and CD40 protein expression induced by either pathogen. Importantly, CD40 expression by osteoblasts was functional, as assessed by ligation of this molecule with recombinant, soluble CD154. CD40 activity was assessed by induction of interleukin-6 and granulocyte-macrophage colony-stimulating factor in osteoblasts following ligation. Cocultures of activated CD4(+) T lymphocytes and osteoblasts could interact via CD40 and CD154, since an antibody against CD40 could block macrophage inflammatory protein-1alpha secretion. Taken together, these studies conclusively demonstrate that infected osteoblasts can upregulate expression of functional CD40 molecules which mediate cytokine secretion. This surprising result further supports the notion that bone-forming osteoblasts can directly interact with CD154-expressing cells (i.e., T lymphocytes) and can contribute to the host response during bone infection.     

Granulocyte-macrophage colony-stimulating factor modulates tapasin expression in human neutrophils

Differential display-polymerase chain reaction (DD-PCR) was used to evaluate changes in mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) treated human neutrophils to better understand how this cytokine affects the functions of neutrophils at the molecular level. Although a variety of cDNA fragments were identified as modulated by GM-CSF with the use of DD-PCR, one fragment in particular, NGS-17 (neutrophil GM-CSF-stimulated fragment #17), was characterized. The NGS-17 fragment hybridized to a 3.8-kh mRNA that encodes for a protein of a predicted molecular mass of 47.6 kDa. After cloning and sequencing, this gene was found to code for the recently sequenced tapasin or TAP-A protein. Immunoprecipitation and immunoblotting studies using anti-tapasin antibodies showed that tapasin is expressed in neutrophils and is associated with the MHC class I-TAP complex. Moreover, tapasin expression was found to be induced by dimethyl sulfoxide and by retinoic acid in HL-60 cells. This is the first report on the expression of tapasin in human neutrophils. It provides novel information, at the molecular level, on how GM-CSF enhances the functions of these cells.     

Altered gene expression in Staphylococcus aureus upon interaction with human endothelial cells

Staphylococcus aureus is isolated from a substantial number of patients with infective endocarditis who are not known to have predisposing heart abnormalities. It has been suggested that the infection is initiated by the direct binding of S. aureus to human vascular endothelium. To determine the mutual response of the endothelial cells and the bacteria, we studied the interaction between S. aureus and human vascular endothelium. Scanning electron microscopic analyses showed that binding of S. aureus to human umbilical vein endothelial cells (HUVEC) mainly occurred via thread-like protrusions extending from the cell surface. Bound bacteria appeared to be internalized via retraction of the protrusions into newly formed invaginations of the endothelial cell surface. The growth phase of S. aureus had a major impact on the interaction with HUVEC. Logarithmically growing bacteria showed increased binding to, and were more readily internalized by, HUVEC compared to stationary-phase bacteria. To assess the bacterial response to the cellular environment, an expression library of S. aureus was used to identify genes whose expression was induced after 4 h of exposure to HUVEC. The identified genes could be divided into different categories based on the functions of the encoded proteins (transport, catabolism, biosynthesis, and DNA repair). Further analyses of five of the S. aureus transposon clones showed that HUVEC as well as human serum are stimuli for triggering gene expression in S. aureus.     

Induction of a neurite-promoting factor in rat brain following injury or deafferentation

Ablation of the entorhinal/occipital cortex in young adult rats caused a several-fold increase in the neurite-promoting activity in extracts of the tissue surrounding the wound and in areas that had been deafferented by the lesion. The time course of induction closely paralleled reactive axon sprouting in the deafferented hippocampus, with maximal levels of neurite-promoting activity reached between 9 and 15 days post-lesion. Aged animals, in which reactive sprouting is deficient, showed no increase in activity by 12 days after deafferentation of the hippocampus. The neurite-promoting activity of brain extracts was non-diffusible, heat-labile, and sensitive to proteolysis. All of the activity bound to diethylaminoethyl (cellulose) and was eluted at 200 mM NaCl. The apparent molecular weight (by gel filtration) of the activity in extracts of uninjured brain was 9-17 kilodaltons, whereas the extracts of injured brain also had peaks or shoulders at 30, 70 and greater than or equal to 200 kilodaltons. These data suggest that the brain neurite-promoting activity resides in one or more proteins. Both the injury-induced and basal activities were different from laminin, nerve growth factor, and polyornithine-bindable neurite-promoting factors. The injury-induced activity was sensitive to repeated freezing and thawing, but this inactivation was reversed by thiol reagents such as glutathione, thioglycerol, and mercaptoethanol. We report a neurite-promoting factor that is induced following brain injury or denervation, and may also be important for reactive axon sprouting after brain injury. The induction of this factor is abnormal in aged animals, as is the reactive sprouting response. The properties of the injury-induced activity distinguish it from the basal activity (found in uninjured brain) and from other characterized neurite-promoting factors.     

Granulocyte-macrophage colony-stimulating factor- and tumor necrosis factor alpha-mediated matrix metalloproteinase production by human osteoblasts and monocytes after infection with Brucella abortus

Osteoarticular complications are common in human brucellosis, but the pathogenic mechanisms involved are largely unknown. Since matrix metalloproteinases (MMPs) are involved in joint and bone damage in inflammatory and infectious diseases, we investigated the production of MMPs by human osteoblasts and monocytes, either upon Brucella abortus infection or upon reciprocal stimulation with factors produced by each infected cell type. B. abortus infection of the normal human osteoblastic cell line hFOB 1.19 triggered a significant release of MMP-2, which was mediated in part by granulocyte-macrophage colony-stimulating factor (GM-CSF) acting on these same cells. Supernatants from infected osteoblasts exhibited increased levels of monocyte chemoattractant protein 1 and induced the migration of human monocytes (THP-1 cell line). Infection with B. abortus induced a high MMP-9 secretion in monocytes, which was also induced by heat-killed B. abortus and by the Omp19 lipoprotein from B. abortus. These effects were mediated by Toll-like receptor 2 and by the action of tumor necrosis factor alpha (TNF-α) produced by these same cells. Supernatants from B. abortus-infected monocytes induced MMP-2 secretion in uninfected osteoblasts, and this effect was mediated by TNF-α. Similarly, supernatants from infected osteoblasts induced MMP-9 secretion in uninfected monocytes. This effect was mediated by GM-CSF, which induced TNF-α production by monocytes, which in turn induced MMP-9 in these cells. These results suggest that MMPs could be potentially involved in the tissue damage observed in osteoarticular brucellosis.     

Induction of apoptosis in myeloid progenitors by granulocyte-macrophage colony-stimulating factor

Spontaneous apoptosis of normal purified bone marrow CD34+ cells induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) via the Fas pathway appears to be mediated by caspase-1 and caspase-8 activity. In seeking an alternative explanation for this observation, the present study examined CD34+ cell growth with different cytokines, cytokine concentrations, caspase inhibitors, cell crowding and different media. Exposure of the normal CD34+ cells to different concentrations of GM-CSF and granulocyte colony-stimulating factor (G-CSF) increased apoptosis at lower concentrations. However, these GM-CSF effects were suppressed by G-CSF. Investigation of the association between apoptosis and crowding and different media showed that: 1) G-CSF and GM-CSF are equally effective as survival factors, and 2) the percentage of apoptotic cells in liquid culture was markedly lower than that found in methylcellulose culture. Finally, immunofluorescence staining showed that Fas was expressed at 10 ng/mL GM-CSF, while Bcl-2 expression was detected at 100 ng/mL. These findings suggest that cytokine concentration, cell culture conditions, cell crowding and cell interactions all are important factors in GM-CSF-induced apoptosis.     

Gene expression of colony-stimulating factors and stem cell factor after myocardial infarction in the mouse

Recent studies have suggested that cytokines such as macrophage colony-stimulating factor (M-CSF) might be involved in the pathogenesis of ischaemic heart disease. Macrophage colony-stimulating factor, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), stem cell factor (SCF), interleukin-3 (IL-3) and interleukin-7 (IL-7) are potent cytokines belonging to the same structual class that may affect function, growth and apoptosis both in the heart and other organs. The aims of the present study were to characterize a post-infarction model in the mouse and to examine mRNA expression of M-CSF, GM-CSF, SCF, IL-3 and IL-7 during the development of heart failure. Myocardial infarction (MI) was induced in mice by ligation of the left coronary artery. Average infarct size was 40% and the mice developed myocardial hypertrophy and pulmonary oedema. Ribonuclease (RNAase) protection assays showed abundant cardiac expression of M-CSF and SCF. After MI, we measured down-regulation of cytokine mRNA expression in the heart (M-CSF, SCF), lung (M-CSF), liver (M-CSF) and spleen (M-CSF) compared with sham. Cardiac G-CSF, GM-CSF and IL-7 mRNAs were not detected. In conclusion, abundant cardiac gene expression of M-CSF and SCF was found. In our mouse model of MI, M-CSF and SCF were down-regulated in the heart and several other organs suggesting specific roles for these cytokines during development of ischaemic heart failure.     

Mechanisms of internalization of Staphylococcus aureus by cultured human osteoblasts

Staphylococcus aureus is an important bone pathogen, and evidence shows that this organism is internalized by chick osteoblasts. Here we report that S. aureus is internalized by human osteoblasts. Internalization was inhibited by monodansylcadaverine and cytochalasin D and to a lesser extent by ouabain, monensin, colchicine, and nocodazole. We propose that internalization occurs via a receptor-mediated pathway, requiring the participation of cytoskeletal elements, principally actin.     

小鼠成骨细胞Wnt信号通路相关因子表达与胰岛素样生长因子1的影响

背景：胰岛素样生长因子1可促进细胞增殖、分化,但其具体作用机制尚不清楚。 目的：观察胰岛素样生长因子1对小鼠成骨细胞增殖﹑分化的影响,及Wnt信号通路相关因子mRNA的表达。 方法：向体外培养的小鼠成骨细胞中加入25 μg/L的胰岛素样生长因子1,分别于培养的第1,2,3,4,5天用CCK-8比色法检测细胞增殖率;于培养的第3,6,9天应用酶联免疫法检测细胞内碱性磷酸酶活性;细胞培养第3天提取总RNA,采用Real-time RT-PCR检测Wnt-3a,低密度脂蛋白受体相关蛋白5,β-catenin mRNA的表达。 结果与结论：25 μg/L胰岛素样生长因子1能够促进成骨细胞增殖,提高碱性磷酸酶活性,而且明显增加成骨细胞中Wnt-3a、低密度脂蛋白受体相关蛋白5、β-catenin mRNA的表达(P < 0.05)。说明胰岛素样生长因子1能够促进成骨细胞的增殖和分化,Wnt信号通路参与了该调控过程。     

Induction of cytokine granulocyte-macrophage colony-stimulating factor and chemokine macrophage inflammatory protein 2 mRNAs in macrophages by Legionella pneumophila or Salmonella typhimurium attachment requires different ligand-receptor systems.

The attachment of bacteria to macrophages is mediated by different ligands and receptors and induces various intracellular molecular responses. In the present study, induction of cytokines and chemokines, especially granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage inflammatory protein 2 (MIP-2), was examined, following bacterial attachment, with regard to the ligand-receptor systems involved. Attachment of Legionella pneumophila or Salmonella typhimurium to cultured mouse peritoneal macrophages increased the steady-state levels of cellular mRNAs for the cytokines interleukin 1beta (IL-1beta), IL-6, and GM-CSF as well as the chemokines MIP-1beta, MIP-2, and KC. However, when macrophages were treated with alpha-methyl-D-mannoside (alphaMM), a competitor of glycopeptide ligands, induction of cytokine mRNAs was inhibited, but the levels of chemokine mRNAs were not. Pretreatment of the bacteria with fresh mouse serum enhanced the level of GM-CSF mRNA but not the level of MIP-2 mRNA. In addition, serum treatment reduced the inhibitory effect of alphaMM on GM-CSF mRNA. These results indicate that bacterial attachment increases the steady-state levels of the cytokine and chemokine mRNAs tested by at least two distinct receptor-ligand systems, namely, one linked to cytokine induction and involving mannose or other sugar residues and the other linked to chemokine induction and relatively alphaMM insensitive. Furthermore, opsonization with serum engages other pathways in the cytokine response which are relatively independent of the alphaMM-sensitive system. Regarding bacterial surface ligands involved in cytokine mRNA induction, evidence is presented that the flagellum may be important in stimulating cytokine GM-CSF message but not chemokine MIP-2 message. Analysis of cytokine GM-CSF and chemokine MIP-2 signaling pathways with protein kinase inhibitors revealed the involvement of calmodulin and myosin light-chain kinase in GM-CSF but not MIP-2 mRNA induction, adding further evidence that several distinct receptor systems are engaged during the process of bacterial attachment and induction of cytokines and chemokines, such as GM-CSF and MIP-2, respectively.     

Tissue factor expression during human and mouse development.

In the adult organism the cellular distribution of tissue factor (TF) expression corresponds to biological boundary layers forming a hemostatic barrier ready to activate blood coagulation after tissue injury. Whether TF expression might also play a role in development is unknown. To determine the significance of TF in ontogenesis, we examined the pattern of TF expression in mouse development and compared it with the distribution of TF in human post-implantation embryos and fetuses of corresponding gestational age. At early embryonic periods of murine (6.5 and 7.5 pc) and human (stage 5) development, there was strong expression of TF in both ectodermal and entodermal cells. In situ hybridization and immunohistochemistry demonstrated that TF mRNA and protein were expressed widely in epithelial areas with high levels of morphogenic activity during organogenesis. Staining for TF was seen during ontogenetic development in tissues such as epidermis, myocardium, bronchial epithelium, and hepatocytes, which express TF in the adult organism. Surprisingly, during renal development and in adults, expression of TF differed between humans and mice. In humans, maturing stage glomeruli were stained for TF whereas in mice, TF was absent from glomeruli but was present in the epithelia of tubular segments. In neuroepithelial cells, there was a substantial expression of TF. Moreover, there was robust TF expression in tissues such as skeletal muscle and pancreas, which do not express it in the adult. In contrast, expression of the physiological ligand for TF, factor VII, was not detectable during early stages of human embryogenesis using immunohistochemistry. The temporal and spatial pattern of TF expression during murine and human development supports the contention that TF serves as an important morphogenic factor during embryogenesis.     

建立小鼠成骨与破骨细胞相互作用的共育新体系

背景：众所周知,骨重建是骨组织中重要的生物学反应过程,其中成骨细胞与破骨细胞发挥了关键作用。但目前,关于骨重建中成骨与破骨细胞间信号传递的深层机制还不清楚。 目的：利用transwell技术,在体外建立一种成骨与破骨细胞的新型共育体系,为深入研究骨重建中成骨与破骨细胞的相互作用提供成熟的实验模型。 方法：采用MC3T3-E1成骨样细胞株与RAW264.7破骨前体细胞株,进行体外成骨与破骨细胞的诱导分化,并利用Transwell共培养板(0.4 µm聚酯膜)建立成骨与破骨细胞的共育体系。共培养6 d后,通过测定细胞活性和碱性磷酸酶(ALP)活力分析成骨细胞的增殖和分化活性,利用抗酒石酸酸性磷酸酶(TRAP)染色、甲苯胺蓝(TB)染色、TRAP活性测定及扫描电镜技术观察破骨细胞的分化及骨吸收功能。 结果与结论：共培养体系中成骨样细胞的无限增殖能力减弱,而分化活性明显增强,同时破骨前体细胞被诱导分化为成熟的破骨细胞,并具有一定的骨吸收功能。因此,该共培养体系可用于骨重建中成骨与破骨细胞间信号通路的深层研究。     

Induction of Per1 expression following an experimentally induced epilepsy in the mouse hippocampus

The Period1 (Per1) is a clock-oscillating gene product that plays an essential role in the generation and modulation of circadian rhythm in the suprachiasmatic nucleus (SCN) of hypothalamus. However, Per1 is also expressed in many other brain regions including cerebral cortex, hippocampus, and amygdala, suggesting that Per1 may be involved in the broader cellular functions in addition to the rhythm regulation. In this study, we found that chemical or electrical seizure-inducing stimulations regulate Per1 expression. Treatments with electric convulsive shock (ECS) or kainic acid (KA) robustly up-regulated the expressions of per1 mRNA and protein in the hippocampal formation and cerebral cortex. In consistent, we found that neuronal depolarization or KA treatment increased per1 mRNA expression in cultured primary cortical neurons. Because it has been demonstrated that Per family molecules contribute to the regulation of stress-induced cell death, we also explored the effect of Per1 overexpression on the survival of cultured neurons. However, neither basal, staurosporine- nor KA-induced neuronal death was affected by forced expression of Per1. Collectively, these results suggest that the Per1 expression is neuronal activity- and epileptogen-dependent, although its functional significance is remained to be explored.     

Compartmentalized transgene expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in mouse lung enhances allergic airways inflammation

To investigate the role of GM-CSF in asthmatic airways inflammation, we have targeted GM-CSF transgene to the airway cells in a mouse model of ovalbumin (OVA)-induced allergic airways inflammation, a model in which there is marked induction of endogenous IL-5 and IL-4 but not GM-CSF. Following intranasal delivery of a replication-deficient adenoviral gene transfer vector (Ad), transgene expression was found localized primarily to the respiratory epithelial cells. Intranasal delivery of 0.03 × 10⁹ plaque-forming units (PFU) of AdGM-CSF into naive BALB/c mice resulted in prolonged and compartmentalized release of GM-CSF transgene protein with a peak concentration of ≈ 80 pg/ml detected in bronchoalveolar lavage fluid (BALF) at day 7, but little in serum. These levels of local GM-CSF expression per se resulted in no eosinophilia and only a minimum of tissue inflammatory responses in the lung of naive mice, similar to those induced by the control vector. However, such GM-CSF expression in the airways of OVA-sensitized mice resulted in a much greater and sustained accumulation of various inflammatory cell types, most noticeably eosinophils, both in BALF and airway tissues for 15–21 days post-OVA aerosol challenge, at which times airways inflammation had largely resolved in control mice. While the levels of IL-5 and IL-4 in BALF and the rate of eosinophil apoptosis were found similar between different treatments, there was an increased number of proliferative leucocytes in the lung receiving GM-CSF gene transfer. Our results thus provide direct experimental evidence that GM-CSF can significantly contribute to the development of allergic airways inflammation through potentiating and prolonging inflammatory infiltration induced by cytokines such as IL-5 and IL-4.     

Gene expression of macrophage colony-stimulating factor and its receptor in human placenta and decidua.

Macrophage colony-stimulating factor (M-CSF) induces proliferation of monocyte/macrophage progenitor cells and can also activate some functions of mature cells including fetally derived placental cells. To study the role of M-CSF in the pregnant female reproductive tract, the expression of M-CSF mRNA and its receptor, c-fms proto-oncogene, in human placenta and decidua was identified. M-CSF and c-fms mRNAs, 4.7Kb and 3.9Kb respectively, were detected by Northern blotting in the early stage placenta and subsequently increased during pregnancy. These mRNAs were not detected in the nonpregnant endometrium but were strongly induced in maternal decidua with the same mRNA size as in the placenta. Northern blot hybridization on the endometrium of a pseudopregnant uterus revealed that the expression of endometrial M-CSF and c-fms mRNAs is regulated by synergistic action of female sex steroid hormones. These findings indicate that, in an autocrine and/or paracrine manner, M-CSF is deeply involved in the local proliferation and differentiation of cells at the materno-fetal interface, and support the placental immunotrophism hypothesis.     

Elevated adenosine cyclic 3'',5''-monophosphate levels in human macrophages following their interaction with Salmonella typhimurium

The mechanisms by which salmonella species establish themselves as facultative intracellular parasites are incompletely understood. Salmonella typhimurium, strain TML, has been shown to elevate adenosine cyclic 3′, 5′-monophosphate (cAMP) levels in rabbit ileum. Since pharmacological elevation of cAMP content is inhibitory to many phagocytic cell functions, we sought to determine if S. typhimurium strain TML would induce elevation of cAMP in human monocyte-derived macrophages and thereby establish itself as a facultative intracellular organism. S. typhimurium strain SL does not affect cAMP levels in the rabbit ileum and served as a control organism. Both strains were used to infect human macrophages and the cAMP content of the macrophages measured by radioimmunoassay. Macrophages infected with strain TML had significantly (P<0.025) higher levels of cAMP at 4, 10 and 24 hr after infection, 19.3±2.5, 17.6±2.4 and 15.4±1.7 pmol per mg protein respectively, than either uninfected macrophages or macrophages infected with strain SL. The cAMP content of SL-infected macrophages did not differ from that of uninfected macrophages. Quantification of intracellular bacteria over 48 hr revealed that strains TML and SL were ingested and killed to the same degree. Furthermore, the 7–10 day old human macrophages used in this study eliminated over 99% of the ingested organisms within 24 hr. Thus, S. typhimurium TML was capable of inducing significant and sustained elevation of cAMP within human macrophages but was not capable of survival within this cell under the in vitro conditions of this study.